Glycolipids are components of a plasma membrane, and various molecular species of glycolipids exist according to the differences of the kinds, number and combining manners of sugar components as well as are distributed specific to species, organs and cells. It has been clarified that glycolipids take an important role related to the growth and differentiation control and to the cellular interaction, and that glycolipids function as receptors of bacteriotoxins, hormones and others and as immunological determinants such as blood group substrates. In addition, some glycolipids have been shown to be tumor antigens due to qualitative and quantitative changes in their composition in association with cell canceration.
Other glycolipids work as regulators of the cell growth mechanism through a growth factor and protein kinase. These instances suggest that the composition change of glycolipids can directly relate to the carcinogenesis mechanism.
On the other hand, establishment of a cell line which produces homogeneous antibodies specific to a single antigenic determinant was reported by Milstein et. al. (Nature, 256, 495-497 (1975)), enabling qualitative and quantitative analyses of substances existing in a very small amount. By using this technique to detect tumor antigens, a number of monoclonal antibodies have been prepared, some of which have been proved to recognize sugar chains of glycolipids or glycoproteins (J. Natl. Cancer Inst. 71, 231-251, (1983)).
For example, monoclonal antibodies against human melanoma, which react with glycolipids such as GD.sub.2 ganglioside or GD.sub.3 ganglioside, have been obtained. Monoclonal antibody NSl9-9, specific to pancreas cancer, reacts with glycolipids having a sugar chain of sialosyl Lewis A type. These antibodies are useful for cancer diagnosis and observation in prognosis and have been studied for use in therapy. The qualitative and quantitative changes of glycolipids are resulted from changes of glycosyltransferase activity in the sugar chain biosynthesis mechanism due to abnormal DNA manifestation. These changes cause abnormal sugar chain structures which are not present in normal tissues. These sugar structures can be used as tumor markers.
Therefore, the importance and usefulness of glycolipids as tumor antigens and tumor markers have been recognized and expected to be applied in the clinical field such as diagnosis and therapy.
When necessary to discriminate particularly between the kinds of sialic acids in this description hereinafter, such discrimination will be made by attaching the prefix, `N-glycolyl` or `N-acetyl`, to the name of a glycolipid.
Among glycolipids, ganglioside is a general term for the glycolipids which contain one or more sialic acid in their sugar chains. As a sialic acid, N-acetylneuraminic acid and N-glycolylneuraminic acid are common. Sialic acids are extensively detected in various organs, cells and body fluids of animal species, while N-glycolylneuraminic acid has not been found in normal humans or chickens.
Heterophile antibodies which are found in patients with serum diseases and agglutinate erythrocytes of sheep, horses, pigs, rabbits and guinea pigs are called H-D antibodies. The antigens recognized by the H-D antibodies are called H-D antigens. That some gangliosides which contain N-glycolylneuraminic acid have the H-D antigen activity was reported (Biochem. Biophys. Res. Commun. 79. 388-395 (1977)), and a sugar structure, NeuGc.alpha.2-3 Gal- was identified a the chief antigen determinant.
Recently, it was reported that an antibody reacting with various gangliosides having the H-D antigen activity was prepared from serum of chickens immunized by N-glycolyl GM.sub.3 ganglioside (II.sup.3 NeuGc-LacCer) having the H-D antigen activity, and that using this antibody N-glycolylneuraminic acid was proved to be present characteristically in human cancer tissues (Biken J. 25, 47-50 (I982)). In addition, several kinds of H-D antigen active gangliosides containing N-glycolylneuraminic acid were detected in the glycolipids extracted from human colon cancer tissues, and glycoproteins containing a H-D antigen-active sugar chain were detected in teratoma tissues (Gann, 75, 1025-1029 (1984)). The H-d antigen-active glycolipids which were detected in human colon cancer tissues were identified as N-glycolyl GM.sub.2 ganglioside, N-glycolyl GM.sub.3 ganglioside, O-acyl-N-glycolyl GM.sub.3 glanglioside and IV.sup.3 NeuGc-nLcOse Cer by using antibodies prepared from chickens, and these glycolipids were not detected in normal tissues (Cancer Res. 45, 3796-3802 (1985)). In a cell line of Marek's disease, lymphoma of chickens, gangliosides having the H-D antigen activity were detected (J. Biochem. (Tokyo), 95, 785-794 (1984)).
Because N-glycolylneuraminic acid and sugar chains containing N-glycolylneuraminic acid are considered as tumor-associated antigens, it is of great importance for cancer diagnosis to detect these acid and sugar chains with high sensitivity and precision.
In order to detect efficiently N-glycolylneuraminic acid, immunoassays are considered to be superior in view of the sensitivity of detection and the accuracy.
Antibodies reacting with a sugar chain which contains N-glycolylneuraminic acid having the H-D antigen activity have been obtained from serum of chickens immunized by purified glycolipid antigens (Molec. Immunol., 19. 87-94 1982)
Polyclonal antibodies having a high specificity to N-glycolyl GM.sub.2 ganglioside can be obtained by immunizing a chicken against N-glycolyl GM.sub.2 ganglioside. (Biochemical and Biophysical Research Communications, Vol. 129, pp. 334-341, 1985). Polyclonal antibodies having a reactivity with N-glycolyl GM.sub.2 ganglioside and N-glycolylGM.sub.3 ganglioside can be obtained by immunizing a chicken against N-glycolyl GM.sub.3 ganglioside. (Cancer Research, Vol. 45 pp. 3796-3802, 1985)
This method, however, has several drawbacks; specifically (1) a large number of purified antigens are required each time the antiserum is needed, (2) the antiserum varies in affinity and titer due to mainly individual differences of immunized animals, (3) for the purification of the desired antibodies, tedious and time-consuming procedures are necessary because of inclusion of undesired antibodies, (4) the shortage of the quantity of the antiserum prepared one time. Thus, in order to carry out an immunoassay with accuracy and the most effectiveness, it has been desired that homogeneous antibodies with stable quality and no inclusion of the other antibodies are able to be supplied in a large quantity. Such manufacturing method of antibodies has been reported as the monoclonal antibody producing technique.
As for monoclonal antibodies specific to sugar chains containing N-glycolyneuraminic acid which have the H-D antigen activity as well as hybridomas having the ability of producing the said antibodies, however, manufacturing have not been reported.
On the other hand, there has been a report that a human monoclonal antibody capable of specifically recognizing melanoma obtained by transforming the lymphocytes of a melanoma patient with EB virus, such human monoclonal antibody reacts with GM.sub.2 ganglioside. (Proceedings of the National Academy of Sciences of the U.S.A.. 80, pp. 5392-5396, 1985).
There has also been another report that human erythro-leukemia cells deposit GM.sub.2 ganglioside threrein, despite of the fact that almost no GM.sub.2 ganglioside is present in normal erythrocytes. (Blood, 62, pp. 1230-1241, 1983).
There has further been a disclosure that, when making use of monoclonal antibody capable of recognizing N-acetyl GM.sub.2 ganglioside and N-glycolyl GM.sub.2 ganglioside each obtained by immunizing mouse melanoma GM.sub.2 ganglioside is manifested mainly in cancerous cells derived from neuroectoderm. (Cancer Research, 46, pp. 4116-4120, 1986).
Accordingly. N-acetyl GM.sub.2 ganglioside and N-glycolyl GM.sub.2 ganglioside are regarded as tumor associated antigens and it is essential from the viewpoint of cancer diagnoses to detect these gangliosides with high sensitivity and accuracy.
As mentioned above, N-glycolyl GM.sub.2 ganglioside, its related glycolipids and sugar chain structures are substantially essential as tumor associated antigens. Therefore. monoclonal antibodies having a reactivity with N-glycolyl GM.sub.2 ganglioside, such as those having a reactivity with N-glycolyl GM.sub.2 ganglioside and N-acetyl GM.sub.2 ganglioside and those having a specific reactivity with N-glycolyl GM.sub.2 ganglioside and other glycolipid-containing N-glycolyl neuraminic acid, are very useful for the diagnosis of various diseases and especially for the diagnoses of human cancers by way of the diagnoses of organs and cells, blood and urine, imaging, or the like. These monoclonal antibodies are also applicable to a missile therapy of bonding a drug to an antibody, or to a therapy utilizing a cytotoxicicity, and they are further applicable to the diagnoses of detecting an antibody against a sugar chain. In the fundamental studies of the relation between a sugar chain and a carcinogenesis mechanism, the structures of sugar chains and glycolipids or the in vivo functional roles thereof, and so forth, various monoclonal antibodies having a reactivity with N-glycolyl GM.sub.2 ganglioside can be used as a useful means.
In this connection, it may be considered to be essential that a monoclonal antibody capable of recognizing at least N-glycolyl GM.sub.2 ganglioside is manufactured.
However, there are the known facts that N-glycolylneuraminic acid is present in the bodies of many kinds of animals including mice but human and chicken, and that N-glycolylneuraminic acid-containing glycolipids including N-glycolyl GM.sub.2 ganglioside which is the subject of the invention, are spread into the system of mice. These glycolipids are the autoantigens of their own. The immunogenicity thereof has been considered to be very weak and, therefore, in the conventional methods using normal mice such as Balb/c mouse and so forth as immune animals, it has been very difficult to obtain a monoclonal antibody capable of producing hybridomas against glycolipids containing N-glycolylneuraminic acid, including N-glycolyl GM.sub.2 ganglioside.
On the other hand, it has been known that animals having an autoimmune disease produce an antibody against an auto-antigen such as an anti-nuclear antibody, antierythrocyte antibody or the like, as described in Immunological Review, Vol. 55, pp. 121-154, 1981. The present inventors devised a method that animals having an autoimmune disease such as, particularly, mice having autoimmune disease, are sensitized to an immunity and, thereby, an immunity reactivity may be increased against N-glycolylneuraminic acid containing glycolipids which may be considered to be autoantigens, and hybridoma may be produced. The inventors tried the method and then achieved the object thereof.
In the course of trying the method, the inventors found that the hybridoma can readily be prepared so as to produce a monoclonal antibody capable of recognizing, particularly. N-glycolyl GM.sub.2 ganglioside, so that this invention can be achieved.